This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

D-Aspartic acid (D-Asp) and nitric oxide (NO) play an important role in tuning testosterone
production in the gonads of male vertebrates. In particular, D-Asp promotes either
the synthesis or the release of testosterone, whereas NO inhibits it. In this study,
we have investigated for the first time in birds the putative effects of D-Asp and
NO on testicular testosterone production in relation to two phases of the reproductive
cycle of the adult captive wild-strain mallard (Anas platyrhynchos) drake. It is a
typical seasonal breeder and its cycle consists of a short reproductive period (RP)
in the spring (April-May) and a non reproductive period (NRP) in the summer (July),
a time when the gonads are quiescent. The presence and the localization of D-Asp and
NO in the testis and the trends of D-Asp, NO and testosterone levels were assessed
during the main phases of the bird's reproductive cycle. Furthermore, in vitro experiments
revealed the direct effect of exogenously administered D-Asp and NO on testosterone
steroidogenesis.

Methods

By using immunohistochemical (IHC) techniques, we studied the presence and the distributional
pattern of D-Asp and NO in the testes of RP and NRP drakes. D-Asp levels were evaluated
by an enzymatic method, whereas NO content, via nitrite, was assessed using biochemical
measurements. Finally, immunoenzymatic techniques determined testicular testosterone
levels.

Results

IHC analyses revealed the presence of D-Asp and NO in Leydig cells. The distributional
pattern of both molecules was in some way correlated to the steroidogenic pathway,
which is involved in autocrine testosterone production. Indeed, whereas NO was present
only during the NRP, D-Asp was almost exclusively present during the RP. Consistently,
the high testosterone testicular content occurring during RP was coupled to a high
D-Asp level and a low NO content in the gonad. By contrast, in sexually inactive drakes
(NRP), the low testosterone content in the gonad was coupled to a low D-Asp content
and to a relatively high NO level. Consequently, to determine the exogenous effects
of the two amino acids on testosterone synthesis, we carried out in vitro experiments
using testis sections deriving from both the RP and NRP. When testis slices were incubated
for 60 or 120 min with D-Asp, testosterone was enhanced, whereas in the presence of
L-Arg, a precursor of NO, it was inhibited.

Conclusion

Our results provide new insights into the involvement of D-Asp and NO in testicular
testosterone production in the adult captive wild-strain mallard drake. The localization
of these two molecules in the Leydig cells in different periods of the reproductive
cycle demonstrates that they play a potential role in regulating local testosterone
production.

Background

It has been long known that testicular testosterone synthesis in vertebrates occurs
in Leydig cells after LH stimulation [1]. In the last few decades, however, several studies have pointed out that in some
laboratory mammals, besides the substantial role of LH [1], a variety of other local biochemical agents are also accountable for testosterone
production. Some of these agents, acting either as stimulators or as inhibitors of
testosterone production, have been localized in Leydig cells and in a variety of neighboring
cells – including Sertoli cells and leucocytes – where they act as autocrine [2], paracrine [3,4] or endocrine hormones [5,6].

Among the array of regulatory agents so far discovered, two molecules, viz, the amino acid D- aspartate (D-Asp) and the inorganic free radical gas nitric oxide
(NO), have lately drawn our attention. These two small molecules belong to the amino
acid matrix generating system. Indeed, whereas D-Asp is the enantiomeric form of L-aspartic
acid (L-Asp) resulting from its conversion via an aspartate racemase, NO is synthesized from L-arginine (L-Arg) via NO synthase (NOS).

Regarding their endocrine role, in the adult rat testis D-Asp operates on Leydig cell
steroidogenic pathway by favoring testosterone synthesis [7-12], whereas NO appears to inhibit it [3,13-15]. These findings agree with a recent study showing that in boar testis testosterone
production in vitro is stimulated by D-Asp and inhibited by an NO generating system [16].

In addition to these previous studies on mammals, a clear definition of the dynamic
relationships between these regulatory factors and testicular testosterone production
has also been obtained by studying seasonally breeding vertebrates, since in these
animals the androgen synthesis is usually restricted to a short reproductive period
of the annual sexual cycle. Indeed, in the green frog Rana esculenta [17] and in the lizard Podarcis sicula [18], highest testosterone production occurs in the springtime breeding period, a time
when D-Asp concentration levels rise in the animals' testes. Moreover, it has been
reported that when exogenous D-Asp is administered to sexually inactive (non reproductive)
adult males, it temporarily induces a significant increase in testosterone output
[17,18]. This field of research, however, has not yet been investigated in birds.

Hence, on the basis of this insightful research, we have investigated the trends of
D-Asp and NO levels, together with testosterone content, in the testes of a typical
seasonal breeder, the duck Anas platyrhynchos, whose male gonads produce both gametes and sex hormones almost exclusively during
the spring [19,20]. Therefore, we determined D-Asp, NO, and testosterone concentrations in the testes
of reproductive (RP) and non reproductive (NRP) adult male ducks. Furthermore, since
the intratesticular source of both D-Asp and NO is still poorly defined, we also investigated
the putative storage and/or production sites of these substances by immunohistochemical
analysis. Finally, experiments in vitro revealed the direct effect of exogenously administered D-Asp and NO on testosterone
steroidogenesis.

We hope that the results of the present study, in addition to having deepened our
understanding of the complex mechanisms involved in the reproductive system of vertebrates,
may also make a significant contribution to the already enlightening data on breeding
biology.

Methods

Reproductive aspects of the sexual cycle in male Anas platyrhynchos

The annual reproductive cycles in male mallards have been well characterized [20-22] on the basis of studies which have compared the relationship between reproductive
features and blood testosterone levels in drakes. In mallards exposed to natural lighting,
the breeding season is restricted to the spring. During winter, birds become photosensitive
and in this state respond to increasing photoperiods with gonadal growth and increasing
plasma testosterone levels [22]. When the breeding season begins, ie, in April and May [21,23], circulating testosterone reaches peak concentrations and the testes become fully
developed for the reproductive activity. On the contrary, in the late spring and early
summer, birds become photorefractory, as the reproductive activity cannot be maintained
throughout the lengthy daylight hours accompanying this season. This response gives
rise to a rapid decline in circulating concentrations of testosterone and regression
of the testes [23]. The decline in plasma concentrations of testosterone is correlated with the postnuptial
molt at the end of which drakes are rendered flightless for 2–3 weeks owing to the
loss of their primary flight feathers [24]. After the postnuptial molt, the drake can no longer be distinguished from the hen
on the basis of plumage color, as the drake's characteristic green head feathers are
replaced by brown feathers. When the growth of new flight feathers is completed, the
prenuptial molt begins [24]. The testes appear to be completely regressed by the time the complete prenuptial
plumage is acquired [24] and the drakes have returned to their characteristic breeding plumage. A second peak
in testosterone occurs in the late fall, but unlike the first, it is not accompanied
by an increase in testicular size; rather, it is thought to be related to pair bond
formation [21].

Animal compliance

Adult male ducks, Anas platyrhynchos, weighing 2.5–3.5 Kg, were obtained from a farmer during their reproductive (RP)
and non reproductive periods (NRP). The collection of samples for the RP was done
between April 29th and May 2nd of 2007. All drakes were in nuptial plumage and no signs of molt were visible. Therefore,
all males were considered to be in their breeding condition. The collection of samples
for the NRP was done in July (15–20, 2007) when the animals showed the typical feature
of post-nuptial molt and the characteristic green head feathers had been replaced
by brown feathers.

Six specimens for each period were utilized. The birds were first anesthetized by
an intramuscular injection of ketamine chloridate (25 mg/kg), and, then sacrificed.
The methods of dissection were in accordance with the Italian guidelines for animal
handling (D. L.vo 116/92) and authorized by the local Animal Care Committee (Servizio
Veterinario della A.S.L. 44, Prot. Vet. 22/95).

The testes were rapidly dissected out. For each testis, three samples were immediately
frozen in liquid nitrogen for biochemical determination of D-Asp, NO and testosterone.
Other samples were first fixed in Bouin's fluid for 12–24 h at room temperature. Then
they were dehydrated, embedded in Paraplast and cut on microtome into 7 μm sections
for the routine histology and immunohistochemical staining of D-Asp and NO. In addition,
fresh testes were utilized for in vitro tests (see below).

Immunohistochemistry

To localize D-Asp and NO in the testes, five randomly chosen sections for each duck
testis were used. The avidin-biotin complex (ABC) (Vector Laboratories, PK-6101) was
used for immunohistochemical analysis. Polyclonal antibodies, raised in rabbits and
directed against D-Asp (Inst. Bas. Sci., Oslo) and nNOS (S. Cruz Biotechnol., SC 648),
were diluted at 1:400 and applied to the sections which had previously been incubated
overnight in a humid chamber at 4°C. The secondary antiserum, raised in goat, and
the PAP complex were applied to the sections for 30 min at room temperature. The sites
on the immunoreactive reaction were amplified using an H2O2 activated solution either of 3,3' diaminobenzidine (DAB) (Vector Laboratories, SK
4100) or of complex DAB-nichel (Vector Laboratories, SK 4100). Sections were counterstained
with hematoxylin, dehydrated in ethanol, cleared in xylene, and mounted.

The specificity of immunostaining for both D-Asp and NO was determined by 1) the substitution
of the primary antiserum with 0.1 M PBS or normal rabbit serum and 2) the overnight
pre-absorption of the primary antiserum with synthetic antigen (for both D-Asp and
NOS, Sigma). These controls never showed any positive reaction.

The preparations were revealed by Nikon E 600 light microscope and microphotographs
were taken by Kodak Tech Pan film.

The scoring of immunoreaction was performed on five randomly chosen sections from
each animal testis on the total of five different fields for each slide.

Morphometry

To test the putative differences of immunopositive cells for D-Asp and NO synthase,
we performed a morphometric analysis using an image analyzer system. Five randomly
chosen sections of testis (D-Asp or NOS immunostained) for each animal of each reproductive
stage were viewed at a magnification of ×1000. The morphological parameter measured
was the number of the immunoreactive Leydig cells in the testis in 1 mm2. Morphometric analysis consisted of digitization of transverse sections viewed under
a Nikon Eclipse E600 light microscope with an attached JVCTK-C1381 photocamera connected
to a Penthium II computer running Lucia ScMeas on Mutech software.

Immunopositive Leydig cells for both D-Asp and NOS between the two reproductive periods
are presented in the form of histograms in Figures 1 and 2, respectively.

Quantitative D-Asp determination

Frozen tissue samples were thawed and homogenized with 0.5 M perchloric acid (PCA)
in a 1:10 ratio and centrifuged at 30,000 g for 20 min. Supernatants, brought to pH
7.5–8.5 with 5 M KOH, were cooled for 30 min at 0°C. Potassium perchlorate precipitate
was removed by centrifugation. Supernatants were adjusted to a pH of about 2.5 with
1 M HCl, and the amino acids were purified on a cation exchange column (AG 50W-X8
resin, hydrogen ionic form, 200–400 mesh, Bio-Rad). Samples were loaded onto columns
(1 × 3 cm) equilibrated with 0.01 M HCl, and, after a wash with 10 ml 0.01 M HCl,
they were eluted with 8 ml of 4 M NH4OH. The elutes were dried by evaporation in small Petri dishes on a hot plate at 40–60°C
under a hood. The dried elutes were dissolved in 1 ml of 0.01 M HCl. They were then
purified by slowly passing them, by means of a syringe, through a Sep-Pak C-18 cartridge
(300 mg; Waters, Milan, Italy) which had been previously activated with methanol or
acetonitrile and washed with distilled water. In order to recover all the amino acids
from these elutes, the cartridge was eluted twice with 2 ml of 0.01 M HCl. The resulting
elutes were combined and either dried using a Savant centrifuge or left to evaporate
in small Petri dishes at 40–50°C under the hood. The dry residues were then dissolved
in 200 μl 0.01 M HCl and analyzed for D-Asp content. The assay procedure was identical
to that previously described by Di Fiore et al. [25]. A standard curve was obtained by applying the enzymatic method to D-Asp solutions
of known concentrations.

Biochemical measurement of NO activity

NO was assessed according to Meli et al. [26] by measuring tissue nitrite, an indicator of NO synthesis. Briefly, frozen samples
of testes were thawed and rinsed in cold PBS buffer (0.1 M, pH 7.4). Then they were
weighed and homogenized (1.2, w/v) in the same ice-cold buffer. The suspensions were
centrifuged at 3500 g for 15 min and aliquots of 100 μl of supernatant were mixed
with aliquots of 100 μl of Griess reagent containing 1% sulphanilamide and 0.1% naphthlethylenediamine
in 5% phosphoric acid. The suspensions were first kept at room temperature for 10
min and then their absorbance (550 nm) was measured in a micro-plate reader (Titertek
multiskan MCC). Solutions of sodium nitrite were used as standards. Nitrite concentrations
in tissues were calculated by comparing their OD with those of the standard solutions.
The results were expressed as mmol of nitrite (NO2-) released by one g of fresh tissue.

Testosterone determination

Determination of testosterone levels in the testis extracts was carried out using
an enzyme immunoassay kit (EIA) (Adaltis, Bologna, Italy). The following limits of
detection were observed for testosterone: sensitivity 6 pg, intra-assay variability
5.3 %, inter-assay variability 7.5 %. Testis samples were homogenized in the ratio
of 1:10 (w/v) with distilled water. The homogenate was then mixed vigorously with
ethyl ether (1:10, v/v) and the ether phase was withdrawn after centrifugation at
3000 g for 10 min. Three extractions were performed. Pooled ether extracts were dried
by evaporation and the residue was dissolved in a 0.5 ml sodium phosphate buffer 0.05
M (pH 7.5), containing BSA at a concentration of 10 mg/ml. Finally, it was utilized
for testosterone immunoassay, as previously reported [25]. The rate of testosterone recovery from testis was about 80%.

In vitro tests

Testes were minced and aliquots (500 mg each) were distributed into a multi-well plate
with 3 ml of KRB buffer (Krebs-Ringer bicarbonate buffer (pH 7.4) containing 10 mM
glucose, 100 μM bacitracin, 0.1% ascorbic acid, and 0.1% bovine serum albumin). The
samples were preincubated for 60 min in an atmosphere of 95% 02/5% CO2 with constant shaking at 60 cycles per min at 37°C. Then the medium was substituted
with 3 ml of fresh KRB buffer containing increasing concentrations of either D-aspartic
acid (D-Asp) in a range between 0.5–2.0 mM or L-arginine (L-Arg) in a range between
1.0–2.0 mM (Sigma). The samples were finally incubated for 60 and 120 min. Controls
were incubated in medium alone. At the end of the incubation period, the media were
aspirated and frozen. Testosterone was measured as reported above. The results were
expressed in terms of nanograms (ng) of testosterone released per gram (g) of tissue.
The experiments were performed in triplicate and data were expressed as means ± SD.
Statistically significant differences were set at p < 0.05 and p < 0.01.

Statistical analysis

Data were compared by analysis of variance followed by Duncan's test for multi-group
comparison and Student t test for between-group comparison. All data were expressed as mean ± standard deviations.
The level of significance was taken at P < 0.01 and P < 0.05.

Results

Presence and distribution of D-Asp and NOS

In the examined ducks, the testes had the typical high vertebrate texture with a tight
network of seminiferous tubules, interposed with interstitial cells, ie Leydig cells. These cells were usually well distinguishable because of their larger
volume and roundish shape. In the intertubular spaces, they were isolated or grouped
in clusters with a variable number of components. The morphometric analysis revealed
no statistical difference (p > 0.05) in the number of Leydig cells expressed during
the two distinct phases of reproductive cycle.

In the testis, seminiferous tubules showed active spermatogenesis only in the reproductive
ducks when Sertoli cells were more visible.

The immunohistochemical tests detected D-Asp and NOS positive materials mostly in
Leydig cells. A feeble positive reaction was also noticed in Sertoli cells, in peritubular
cells surrounding the seminiferous tubule, in spermatogonia and in spermatids. No
differences in their staining pattern were observed during either period (description
not given).

Differences in the number of Leydig cells involved and in their staining intensity
to D-Asp and NOS immunoreactive methods were observed in animals deriving from both
phases of reproductive cycle. Indeed, whereas NOS staining intensity was mainly greater
during NRP, D-Asp immunoreactivity was greater during RP.

Figure 1 reports D-Asp immunoreactive distribution in testes of ducks belonging to the RP
(Figure. 1A and 1B) and to the NRP (Figure. 1C). D-Asp immunoreactivity was primarily found in Leydig cells (Figure. 1A and 1B). The positive Leydig cells were more numerous and mainly grouped in clusters during
the RP (Figure. 1A). Figure 1B shows a section at higher magnification of immunopositive Leydig cell types. During
the NRP (Figure. 1C), reactive Leydig cells were present only in few of the examined sections and, when
present, they appeared isolated. The morphometric analysis (Figure. 1D) revealed the quantification of immunopositive Leydig cells/mm2 during both periods: immunopositivity was higher during the RP than during the NRP
(p < 0.01).

NOS immunoreactive distribution in the testes of ducks belonging to the RP (Figure.
2A) and to the NRP (Figure. 2B and 2C) is shown in Figure. 2. In the latter, NOS-positive Leydig cells were always present and the finding of
interstitial spaces entirely filled with reactive Leydig cells was common. Figure
2B shows a section at higher magnification of immunopositive Leydig cell types. Figure
2A shows a sample section of the RP, where the immunopositive Leydig cell types were
weak. Figure 2D shows the histograms of the quantification of immunopositive Leydig cells/mm2 during both periods: they were higher during the NRP than during the RP (p < 0.01).

Figure 2.Representative immunohistochemical distribution and localization of NO synthase in
the testes of duck A. platyrhynchos during the reproductive period (RP) (A) and the non reproductive period (NRP) (B and C). NOS positive cytotypes were found elsewhere in seminiferous tubules, but they were
very rare in the interstitial tissue of RP (A). Immunopositivity was mainly localized in Leydig cells of the NRP (B and C). Histograms represent quantification of immunopositive Leydig cells/mm2 (D). **, p < 0.01 NRP versus RP. L, lumen. Scale bars: a, 100 μm; b, 50 μm; c, 20 μm.

Determination of testicular D-Asp, NO activity and testosterone

Figure 3 reports the levels of D-Asp, NO and testosterone in the duck testicular extracts
of the RP and the NRP. Biochemical analysis revealed that D-Asp concentration in duck
testis was higher during the RP than during the NRP (23.1 ± 2.9 nmol/gr of wet tissue
and 11.0 ± 1.2 nmol/gr of wet tissue, respectively), (p < 0.01).

Figure 3.Concentrations of D-Asp, NO levels and titers of testosterone in the testes of A. platyrhynchos during the reproductive period (RP) and non reproductive period (NRP). D-Asp concentration was higher in RP, corresponding to maximum testosterone titre
and to minimum NO level. Each point represents the mean value ± S.D. from six individual
ducks. **, p < 0.01, Reproductive versus Non Reproductive period.

In vitro effect of D-Asp and L-Arg on testosterone release

To verify the exogenous effects of D-Asp and L-Arg, a precursor of NO, on testicular
testosterone release, we incubated slices of duck testes for 60 and 120 min in a medium
containing either D-Asp or L-Arg (Figure 4). As expected, D-Asp induced a significant increase in testosterone, whereas L-Arg
caused a decrease. Specifically, after a 60-min incubation with D-Asp, testosterone
synthesis was higher than that of control (2.0 mM [p < 0.01] and 1.0 mM [p < 0.05]).
Similarly, after 120 minutes of incubation, the increases were significantly higher
than those of control (1.0 mM and 2.0 mM D-Asp concentration [p < 0.01]). Furthermore,
when the exogenous stimulation was absent, we noted that testosterone production at
120 min was approximately 2-fold higher than that at 60 min. In fact, although a 60-min
incubation with D-Asp yielded higher testosterone values, the proportionate increase
at 120 min appeared approximately identical to that of control.

Figure 4.Effect of increasing concentrations of D-Asp (1.0–2.0 mM) and of L-Arg (1.0–2.0 mM)
on testosterone release from testes of A. platyrhynchos. The testis slices were incubated for 60 (□) or 120 (■) min. Testosterone levels were
enhanced after 60 or 120 min incubation with D-Asp, whereas they were inhibited in
the presence of L-Arg. The level of significance was taken at p < 0.05 (*) and p <
0.01 (* *) versus respective controls.

Incubations with L-Arg yielded a decrease in testosterone levels after 120 min at
1.0 mM (p < 0.05) and after 60 and 120 min of incubation at 2.0 mM (p < 0.01), as
compared to controls.

Discussion

Since the 1950s, Leydig cells have been identified as the androgen source in vertebrate
testes, and, thereafter, in the late 1960s and early 1970s, their endocrine activity
was shown to be strictly dependent on the pituitary LH [1]. In the last decades, however, new technical advances have made it possible to delve
deeper into the intricate mechanisms underlying the regulation of steroidogenesis,
particularly in mammalian testes. These studies have soon shown that a myriad of intratesticular
agents present in Leydig cells can alter testosterone production by acting as autocrine
[2], paracrine [3,4], and endocrine [5,6] excitatory or inhibitory factors.

In recent years, several studies in this burgeoning filed of research have attributed
a great importance to the regulating properties of two molecules, viz, the amino acid D-aspartic acid (D-Asp) and the free radical Nitrogen Oxide (NO)
in the reproductive systems of mammals [27,28].

However, the putative role of D-Asp and NO in testosterone production in the male
gonads of some seasonal breeder vertebrates also offers a good basic unmanipulated
model to improve our understanding of reproductive biology. In these animals, the
gonads are active only for a relatively short period each year, specifically in the
spring. During this season, sex hormone plasma concentrations peak, gametes are produced
and the secondary sexual characters (SSC) completely develop. By contrast, sex hormone
production is very low throughout the rest of the year.

In the present study we report for the first time the involvement of D-Asp and NO
in the testis steroidogenesis of the mallard A. platyrhynchos, a typical seasonal breeder. Such hypothesis was verified by assaying testicular
testosterone production in relation to the presence and distribution of D-Asp and
NO during the reproductive period (RP) and the non reproductive period (NRP). Moreover,
to better evaluate the role of exogenous D-Asp and NO in testicular testosterone synthesis,
in vitro experiments were carried out.

Immunocytological techniques have allowed scientists to localize D-Asp in Leydig and
Sertoli cells [7], as well as in peritubular and several germinal epithelium elements [29]. A strong correlation between D-Asp content and testosterone synthesis has been found
in the testes of rats [7-12], boar [16], amphibians [17], and reptiles [18], where, indeed, D-Asp stimulates testosterone synthesis and release. In Leydig cells,
D-Asp appears to act on genes since it induces the synthesis of the regulatory protein
StAr involved in the synthesis of testosterone [11]. Consistently, in the present study we found that free D-Asp was endogenously contained
in the mallard testis and varied throughout the two sexual reproductive cycles, ie the reproductive period (RP) and non reproductive period (NRP). D-Asp concentration
was higher during the RP (23.9 ± 2.9 nmol/g tissue) than during the NRP (11.0 ± 1.2
nmol/g tissue). Such discrepancy was also evident in IHC studies, in which, although
no difference in the number of Leydig cells was found between the two phases of the
reproductive cycle considered, a significant increase in D-Asp immunopositive Leydig
cells was detected during the RP but not during the NRP.

Circumstantial evidence suggests that Nitric Oxide (NO) is normally synthesized in
the mammalian testis. The enzyme nitric oxide synthase (NOS), which determines NO
formation in tissues through the oxidation of L-arginine, has indeed been localized
in several testicular components, including Leydig and Sertoli cells [13,16,30-33]. Intriguingly, our immunohistochemical observations only in part support such theories,
for we found that NO was mostly localized in Leydig cells. Intriguingly, here we report
for the first time that NO is far more expressed during the NRP than during the RP.
Indeed, our morphometric analysis revealed a more intense NO-immunopositivity during
the NRP, as compared to the RP. Moreover, NO concentration resulted inversely correlated
to either D-Asp or testosterone in the two periods of the reproductive cycle. In fact,
the highest NO concentrations were registered in the testes during the NRP (84.7 ±
11.8 nmol/g tissue). Interestingly, this increase was paralleled by minimum testosterone
levels (0.87 ± 0.2 ng/g tissue) and by the regression of the testes. Thus considering
that during this period the animal undergoes postnuptial molt [21], we could advance the hypothesis that highest levels of NO are somehow related to
molt. Indeed, a recent study done on Gallus domesticus has emphasized that NO production increases after molt induction [34]. Nonetheless, further studies are still required to validate this hypothesis in mallards.

In current literature, various functions have been ascribed to NO in the testis, such
as the stimulation of germ cell metabolism/apoptosis, the relaxation of vascular myocytes,
the regulation of peristalsis and permeability of the seminiferous tubule [28], the inhibition of sperm motility, the induction of peritubular myofibroblast contraction
[14,31,35-39], and, lastly, the significant depression of testosterone synthesis in Leydig cells.
The latter mechanism, however, is still poorly defined. So far, it has been speculated
that NO could inhibit Leydig cell testosterone synthesis either directly or indirectly.
More specifically, when NO is released within the testes by either Leydig cells or
by intraorganic nerve fibres [32], it could activate soluble guanylyl cyclase (sGC) and, consequently, cyclic guanosin
monophosphate (cGMP) [30,38]. The latter, in turn, could induce the stimulation of a yet poorly defined cytoplasmic
protein kinase [40]. It is worthy of note that the sGC/cGMP pathway of cellular response to NO has been
proposed not only for the Leydig cell but also for other testicular cytotypes including
Sertoli, endothelial, peritubular, and germ cells [41].

Even though the literature on NO underscores its direct or indirect role in the regulation
of gonadal functions, specific research on avian species is still limited. Only recently,
in an interesting study carried out on Japanese quails, it has been suggested that
NO plays a positive regulatory role in the physiology of gonadal and adrenal axis
and photosexual responses [42]. Remarkably, however, the data that we report in this study, despite appearing inconsistent
with current findings, actually do highlight two different aspects of analysis with
respect to NO involvement. For instance, although the study on sexually immature Japanese
quails pinpoints several factors that can enhance the regulation of hypothalamo-hypophyseal-gonadal
and -adrenal axis, it does not mention how NO could possibly regulate steroidogenesis
in Leydig cells. These discrepancies could be ascribable to the differences among
species, in particular with regard to their status of sexual maturity and/or regulation
machinery considered.

Moreover, what makes this study particularly insightful is the evidence that both
D-Asp and NO displayed a local and direct action on Leydig cells that was strictly
related to the two different phases of the sexual cycle. Indeed, it emerged that the
two phases of the reproductive cycle were characterized by different biochemical pathways.
More specifically, we observed that during the spring, whereas NO content was relatively
low, highest concentrations of testosterone and D-Asp occurred in the testes of sexually
active drakes. Conversely, in the testes of non reproductive males, testosterone and
D-Asp levels declined markedly, whereas NO potential synthesis was relatively high.
These intriguing findings were further corroborated by parallel results obtained from
our in vitro experiments. The testosterone release from testis slices of reproductive ducks was
stimulated when we added exogenous D-Asp to the incubation medium. By contrast, it
was inhibited when we incubated the sections with an NO generating system.

In conclusion, these results provide substantial support to the hypothesis that D-Asp
and NO play important regulatory roles in testicular testosterone production. Thus,
given their intrinsic capability to balance hormonal production in Leydig cells, they
ought to be considered putative opposite regulators of local testosterone synthesis.
Equally important, our findings suggest that these factors could also be implicated
in Leydig cell endocrine activity, as it occurs in seasonally reproducing vertebrates.
Finally, we hope that the results of the present study, in addition to having deepened
our understanding of the complex mechanisms involved in the reproductive system of
vertebrates, may also make a significant contribution to the already enlightening
data on breeding biology.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

MMDF devised the study, organized the planning of the experiments, developed the method
for D-Asp determination, performed in vitro experiments and wrote the manuscript. CL carried out the sampling during the reproductive
stages, developed the IHC methods and NO assays, took part in the discussion of the
results and helped write manuscript. LA measured testosterone levels, made statistical
analyses, took part in the discussion of the results. VB participated in the discussion
of the results and helped write the manuscript. All authors read and approved the
final manuscript.

Acknowledgements

We would like to thank Dr. Paola Merolla for editing the present manuscript.

This work was partly supported by a MIUR grant (cofin PRIN 2004, prot. 2004051955).